1. Field of Invention
This invention relates to treatment of human beings for conditions associated with deficiencies in the N-6-trimethyl-L-lysine (TML) pathway affecting biosynthesis of carnitine. Such conditions include (1) clinical hyperammonemia, (2) clinical state of impairment of carnitine or carnitine esters, (3) decreased fatty acid metabolism, (4) lower energy production or lower ATP production in the body, or (5) clinically high levels of pyruvate.
2. Description of Related Art
All material referenced in the prior provisional and non-provisional applications are hereby incorporated by reference.
Role of L-Carnitine
From a biochemical standpoint, L-carnitine plays an essential role in energy metabolism. In fatty acid metabolism, it serves as shuttle between the mitochondrial membrane and the mitochondria inner-workings permitting breakdown of the long-chain carbon fragment.
The more important role of L-carnitine is in maintaining a balance between the concentrations of a compound called acyl CoA in the cell compartments. For sugar to be metabolized, they are sequentially degraded to smaller fragments until carbon dioxide is produced, and along the way energy is conserved. Acyl CoA is an important intermediate in transfer of energy. Accordingly, it is important that the concentration of Acyl CoA be regulated and this function falls on L-carnitine. The role of L-carnitine and L-carnitine supplementation during exercise in humans has been illustrated (Brass E. P., Hiatt W. R., J. Am. Coll. Nutr., 17(3):207-215, 1998).
It has also been shown that defects in fatty acid oxidation are a source of major morbidity, particularly among children. Fatty acid oxidation defects encompass a spectrum of clinical disorders, including recurrent hypoglycemic, hypoketotic encephalopathy or Reye-like syndrome in infancy with secondary seizures and potential developmental delay, progressive lipid storage myopathy, recurrent myoglobinuria, neuropathy, and progressive cardiomyopathy. (I. Tein, J Child Neurol. 2002 December; 17 Suppl 3:3S57-82; discussion 3S82-3).
Supplementation or treatment of a number of these diseases/disorders with L-carnitine has had beneficial effects. For example, some researchers believe L-carnitine supplementation may complement other therapies for the treatment of AIDS. (Effect of L-carnitine on human immunodeficiency virus-1 infection-associated apoptosis; Moretti S., Alesse E., Di Marzio L., a pilot study. Blood, 91(10):3817-3824, 1998). According to the authors, the treatment of immunodeficiency virus type 1 infections, acquired immune deficiency syndrome (AIDS), may elicit or cause carnitine deficiency problems. Additionally, some epileptic patients may benefit from carnitine supplementation or treatment.
L-carnitine may be essential or “conditionally” essential for several groups of people including: normal infants, premature infants, and both children and adults suffering from a variety of genetic, infectious, and injury-related illnesses. For example, some cardiomyopathies, which afflict children, are due to metabolic errors or deficiencies. There is data that supports treatment of some myocardial dysfunctions with L-carnitine supplementation. (Winter, S., Jue, K., Prochazka J., Francis, P., Hamilton, W., Linn, L., Helton, E. (1995) J. Child Neurol. 10, Supple 2: S45-51.)
L-carnitine may also play an essential role in the treatment of several disease conditions. Administration of L-carnitine prevents acute ammonia toxicity and enhances the efficacy of ammonia elimination as urea and glutamine. In addition, the cytotoxic effects of ammonia, possibly arising from lipid peroxidation, are ameliorated by L-carnitine. These data indicate the feasibility of utilization of L-carnitine in the therapy of human hyperammonemic syndromes, both for reducing the levels of ammonia and preventing its toxic effects. (O'Connor J E, Adv Exp Med. Biol. 1990; 272:183-95).
L-carnitine deficiency can be defined as a decrease of intracellular L-carnitine, leading to an accumulation of acyl-CoA esters and an inhibition of acyl-transport via the mitochondrial inner membrane. This may cause disease by the following processes:
A. Inhibition of the mitochondrial oxidation of long-chain fatty acids during fasting causes heart or liver failure. The latter may cause encephalopathy by hypoketonaemia, hypoglycaemia and hyper-ammonaemia. It was shown that acetyl-L-carnitine fed to old rats partially restores mitochondrial function and ambulatory activity (Hagen T M, Ingersoll R. T, Wehr C. M, Proc. Natl. Acad. Sci. USA., 95(16): 9562-9566, 1998).
B. Increased acyl-CoA esters inhibit many enzymes and carriers. Long-chain acyl-CoA affects mitochondrial oxidative phosphorylation at the adenine nucleotide carrier, and also inhibits other mitochondrial enzymes such as glutamate dehydrogenase, L-carnitine acetyltransferase and NAD transhydrogenase. (Scholte H R, J Clin Chem Clin Biochem. May 1990; 28(5): 35)
C. Accumulation of triacylglycerols in organs increases stress susceptibility by an exaggerated response to hormonal stimuli (lyer, R. N., Khan, A. A., Gupta, A., Vajifdar, B. U., Lokhandwala, Y. Y., J Assoc Physicians India, 8(11):1050-1052, 2000).
D. Effect of L-carnitine on exercise tolerance in chronic stable angina: a multicenter, double-blind, randomized, placebo controlled crossover study (Cherchi, A., Lai, C., Angelino, F., et al., Int. J. Clin. Pharmacol. Ther. Toxicol., 23(10):569-572, 1985).
E. Decreased mitochondrial acetyl-export lowers acetylcholine synthesis in the nervous system.
Primary L-carnitine deficiency can be defined as a genetic defect in the transport or biosynthesis of L-carnitine. Until now, only defects at the level of L-carnitine transport have been discovered. The most severe form of primary L-carnitine deficiency is the consequence of a lesion of the L-carnitine transport protein in the brush border membrane of the renal tubules. This defect causes cardiomyopathy or hepatic encephalopathy usually in combination with skeletal myopathy. In a patient with cardiomyopathy and without myopathy, it was found that L-carnitine transport at the level of the small intestinal epithelial brush border was also inhibited. The patient was cured by L-carnitine supplementation. Muscle L-carnitine increased, but remained too low, suggesting that L-carnitine transport in muscle is also inhibited. L-carnitine transport in fibroblasts was normal, which disagrees with literature reports for similar patients. W. R. Treem, C. A. Stanley, D. N. Finegold, D. E. Hale, P. M. Coates, N. Engl. J. Med, 319, 1331-1336, 1988; G. Karpati, S. Carpenter, A. G. Engel, G. Watters, J., Allen, S. Rothman, G. Klassen, O. A. Mamer, Neurology, 25, 16-24, 1975; B. O. Eriksson, S. Lindstedt, I. Nordin, Eur. J. Pediatr., 147, 662-663, 1988; H. R. Scholte, R. Rodrigues Periera, P. C. de Jonge, I. E. Luyt-Houwen, M. Verduin, J. D. Ross, J. Clin. Chem. Clin. Biochem. 28, 351-357, 1990; C. A. Stanley, S. DeLeeuw, P. M. Coates, C. Vianey-Liaud, P. Divry, J. P. Bonnefont, J. M. Saudubray, M. Haymond, F. K. Tretz, G. N. Breningstall, Ann. Neuro. 30, 709-716, 1991; Y. Wang, J. Ye, V. Ganapati, N. Longo, Proc. Natl. Acad. Sci. USA 96, 2356-23601999.
In summary, L-carnitine plays a critical role in enhancing fat metabolism. Reports attest to the fact that L-carnitine works by transporting fatty acids to be burned for fuel, increasing both energy supply and lean muscle mass. Most reports also indicate that unless an individual is deficient in L-carnitine, it is an unnecessary ergogenic aid. This contrasts with an apparent need in case of L-carnitine deficiency (e.g., in the case pursued by the inventors of Late Infantile Neuronal Ceroid Lipofuscinosis—one form of Batten Disease), of the correct operation of the endogenous production of L-carnitine. This need was corroborated in the observations of dogs with Batten Disease given exogenous L-Carnitine (Siakotos A. N., Hutchins G. D., Farlow M. R., Katz M. L., European Journal of Paediatric Neurology 5 Suppl A: 151-6, 2001) and those of the parents of the child who was afflicted with LINCL (discussed below).
ATP Dependence on Endogenous L-Carnitine
Optimal ATP production from either dietary or stored fatty acids is dependent on L-carnitine. L-carnitine has several roles, most of which involve conjugation of acyl residues to the b-hydroxyl group of the L-carnitine with subsequent translocation of this complex from one cellular compartment to another. Deficiencies in L-carnitine have been implicated in a number of diseases. For example, in CLN3, proteins have been found to cause modulation of the cell growth rates and apoptosis (Persaud-Sawin D. A., Van Dongen A., Boustany R. M., Human Molecular Genetics. II (18):2129-42, 2002). It has been shown that defects in lysosomal enzymes cause Neuronal Ceroid Lipofuscinoses (NCLs), CLN1 and CLN2. (Hofmann S. L., Atashband A., Cho S. K., Das A. K., Gupta P., Lu J. Y., Current Molecular Medicine. 2(5):423-37, 2002). It has been also shown that the conditions of Parkinson's disease are present when there is dysfunction in both striatal and nigral neurons and this dysfunction results in autosomal dominant adult neuronal ceroid lipofuscinosis (Nijssen, P. C., Brusse, E., Leyten, A. C., Martin, J. J., Teepen, J. L., Roos, R. A., Movement Disorders, 17(3):482-7, 2002). It has been shown that abnormal accumulation of specific proteins occurs in the neuronal ceroid lipofuscinosis/Batten disease. These conditions result due to defect in the lysosomal proteases and related enzymes. The phenomenon is commonly termed as lysosomal proteinoses. This abnormal accumulation of proteins in the lysosomes has been shown to be responsible for major diseases such as Alzheimer disease, alpha-synuclein in Parkinson's disease, Lewy body dementia (Gupta, P., Hofmann, S. L., Molecular Psychiatry. 7(5):434-6, 2002). An autoantibody inhibitory to glutamic acid decarboxylase in the neurodegenerative disorder, Batten disease has been reported (Chattopadhyay, S., Ito, M., Cooper, J. D., Brooks, A. I., Curran, T. M., Powers, J. M., Pearce, D. A., Human Molecular Genetics. 11(12): 1421-31, 2002). Mutations in different proteins result in similar diseases of neuronal ceroid lipofuscinoses (Weimer, J. M., Kriscenski-Perry, E., Elshatory, Y., Pearce, D. A., NeuroMolecular Medicine. 1(2): 111-24, 2002). Lysosomal localization of the neuronal ceroid lipofuscinosis CLN5 protein. (Isosomppi, J., Vesa, J., Jalanko, A., Peltonen, L., Human Molecular Genetics. 11(8):885-91, 2002).
Carnitine Biosynthesis & Metabolism
N-6-Trimethyl-L-Lysine (TML)
The enzyme Tripeptidyl Peptidase 1 (TPP-1) is responsible for cleaving “protein bound N-6-trimethyl-L-lysine” with the resulting products of free TML and amino acids in normal people. However, in children who have Late-Infantile Neuronal Ceroid Lipofuscinoses (LINCL), the TPP-1 is defective and the “protein-bound TML” is not broken down (M. L. Kaz, Biochem. Biophy. Acta, 1317, 192-198, 1996). It therefore becomes the storage material in the lysosome. Eventually it builds up and then consequently “blows up” the lysosome, causing eventual massive neuronal damage in brain and eventually death (P. Gupta and S. L. Hofmann, Molecular Psychiatry, 7, 434-436, 2002).
It has been shown that there is specific accumulation of a hydrophobic protein, subunit c of ATP synthase, in lysosomes from the cells of patients with LINCL, and is caused by a defect in the CLN2 gene product, TPP-1. The data by the authors show that TPP-1 is involved in the initial degradation of subunit c in lysosomes and suggest that its absence leads directly to the lysosomal accumulation of subunit c (Ezaki, J., Takeda-Ezaki, M., Kominami, E., J Biochem (tokyo) September, 128(3), 509, 2000).
Lysosomal hydrolysis of these proteins results in the release of TML, which is the first metabolite of L-carnitine biosynthesis. Hepatic synthesis of carnitine takes place from protein-bound N-6-trimethyl-L-lysine. Lysosmal digestion of methyl-lysine labeled asialo-fetuin was carried out (LaBadie, J., Dunn, W. A. and Aronson Jr, N. N. Biochem. J. 160, 85-95, 1976). L-carnitine biosynthesis has been studied, such as, from gamma-butyrobetaine and from exogenous protein-bound-6-N-trimethyl-L-lysine by perfused guinea pig liver. In this connection, the effect of ascorbate deficiency on the in situ activity of gamma-butyrobetaine hydroxylase was demonstrated (Dunn, W. A., Rettura, G., Seifter, E. and Englard, S., J. Biol. Chem. 259, 10764-10770, 1984).
TML to L-Carnitine Pathway
TML is first hydroxylated on its 3-position to form 3-hydroxy-N-6-trimethyl-L-lysine (HTML). The aldolytic cleavage of HTML with HTML Aldolase (HTMLA) yields trimethylaminobutyraldehyde (TMABA) and glycine. Dehydrogenation of TMABA by TMABA dehydrogenase (TMABA-DH) results in the formation of 4-N-trimethylaminobutyrate (butyrobetaine). In the last step, gamma-butyrobetaine is hydroxylated on the 3 position by gamma-butyrobetain deoxygenase (BBD; EC to yield L-carnitine (Frederic M. Vaz and Ronald J. A. Wanders, Biochem. J. 361, 417-429, 2000). Very little is known about HTMLA. It might be identical to serine and lycine hydroxymethyltransferase (SHMT) which catalyses the tetrahydrofolate-dependent interconversion of serine and glycine (Girgis, S., Nasrallah, I. M., Suh, J. R., Oppenheim, E., Zanetti, K. A., Mastri, M. G. and Stover, P. J., Gene 210, 315-324, 1998). Purification and characterization of cytosolic and mitochondrial serine hydroxymethyltrasferase from rat liver was carried out (Ogawa, H. and Fujioka, M. J. Biochem. (Tokyo) 90, 381-390, 1981). SHMT also catalyses the aldol cleavage of other beta-hydroxylamino acids in absence of tetrahydrofolate, including HTML (Girgis, S., Nasrallah, I. M., Suh, J. R., Oppenheim, E., Zanetti, K. A., Mastri, M. G. and Stover, P. J. Gene 210, 315-324, 1998). Synthesis of butyrobetaine and L-carnitine from protein bound TML is inhibited by 1-amino-D-proline, an antagonist of vitamin B6. This inhibitory effect of 1-amino-D-proline on the production of L-carnitine from exogenous protein-bound N-6-trimethyl-L-lysine by the perfused rat liver has been shown. (Dunn, W. A., Aronson Jr, N. N. and Englard, S., J. Biol. Chem. 257, 7948-7951, 1982).
It is well known from the biochemistry of the metabolic pathway of TML to HTML that certain cofactors; such as 2-oxoglutarate, Fe2+, molecular oxygen and ascorbate, have to be present. Similarly in the subsequent steps of metabolic pathway from HTML to L-carnitine, the biochemically defined cofactors have to be present. The cofactors (2-oxoglutarate, Fe2+, molecular oxygen, and ascorbate) have been established by a number of researchers during the enzymatic hydroxylation of TML. It is likely that other chemicals will work as cofactors as well. For example, DTT (dithiothreitol) has been used instead of ascorbic acid (which is required to keep Fe2+ in reduced form), in test tube conditions. Besides the aforesaid cofactors, calcium ion was found to cause significant enhancement in the conversion of TML to HTML (D. S. Sachan, C. L. Hoppel, Biochem. J., 188, 529-534, 1980).
4-N-trimethylaminobutyraldehyde dehydrogenase (TMABA-DH) catalyzes the dehydrogenation of 4-N-trimethylamino butyraldehyde to butyrobetaine. TMABA-DH has an absolute requirement for NAD+. In human tissues, the rate of TMABA dehydrogenation is highest in liver, substantial in kidney, but low in brain, heart and muscle (Rebouche, C. J. and Engel, A. G., Biochim. Biophys. Acta, 22-29, 1980). TMABA-DH has been purified from beef liver (Hulse, J. D. and Henderson, L. M., Fed. Proc. Fed. Am. Soc. Exp. Biol., 38, 676, 1979).
Gamma-butyrobetaine dioxygenase (BBD) catalyses the stereospecific hydroxylation of butyrobetaine to L-carnitine in mammalian studies. BBD activity was stimulated considerably by 2-oxoglutarate, and the enzyme requires molecular oxygen, Fe2+ and ascorbate for activity. (Lindblad, B., Lindstedt, G. and Tofft, M., J. Am. Chem. Soc., 91, 4604-4606, 1969). BBD activity has been found to be localized in the cytosol.
Kakimoto and Akazawa were the first to identify TML in human urine. All methods to assay TML in either plasma, urine or tissue samples use the same sample work-up. The concentration of TML in plasma is relatively constant in both human and rat, ranging from 0.2 to 1.3 micromole. Plasma levels of TML have been shown to correlate with body mass. In humans, urinary TML concentration is proportional to that of creatine. Furthermore, TML is not reabsorbed by kidney in humans. (Davis, A. T., Ingalls, S. T. and Hoppel, C. L J. Chromatogr. 306, 79-87, 1984.). In humans, TML concentrations range between 2 to 8 micromole per mmole of creatine. (Kakimoto, Y. and Akazawa, S., J. Biol. Chem. 245, 5751-5758, 1970).
Butyrobetaine is the last step in the synthesis of L-carnitine. The level of butyrobetaine in urine is low (about 0.3 micromole/mmol creatinine) (F. M. Vaz, B. Melegh, J. Bene, D. Cuebas, D. A. Gage, A Bootsma, P. Vreken, A. H. van Gennip, L. L. Bieber and R. J. A. Wanders, unpublished work) compared with the concentration in plasma of 4.8 micromole (Sandor, A., Minkler, P. E., Ingalls, S. T. and Hoppel, C. L., Clin. Chim. Acta., 176, 17-27, 1988).
Factors in the Biosynthesis & Control of L-Carnitine and N-6-Trimethyl-L-Lysine
Major sources of L-carnitine in the human diet are meat, fish and dairy products. Omnivorous humans generally ingest 2-12 micromoles of L-carnitine per day per kg of body weight. This is more than the L-carnitine produced endogenously, which has been estimated to be 1.2 micromole per day per kg of body weight. In omnivorous humans, approximately 75% of body L-carnitine sources come from the diet and 25% come from de novo biosynthesis. Since L-carnitine is present primarily in foods of animal origin, strict vegetarians obtain <0.1 micromole per day per kg of body weight. Strict vegetarians obtain more than 90% of their L-carnitine through biosynthesis.
Two primary intermediates have been proposed as the factors which limit biosynthesis of L-carnitine via their availability. These two intermediaries are g-butyrobetaine and N-6-trimethyl-L-lysine. Studies have shown that increasing the amount of either of these two intermediates in the bloodstream will increase the production of L-carnitine 100-fold in rats and 3-fold in human infants and adults (Olson and Rebouche, J. Nutr. 117(6), 1024-31, 1987). Thus, L-carnitine biosynthesis may be regulated by one or all of the three enzymes which, together, catalyze the transformation of N-6-trimethyl-L-lysine into g-butyrobetaine. The high level of L-carnitine synthesis from exogenous L-carnitine precursors suggests that the enzymatic capacity to synthesize L-carnitine from TML and butyrobetain is much higher than is usually utilized. This suggests that only the availability of TML is the rate limiting step in the regulation of feedback inhibition for L-carnitine biosynthesis (Schematic 1). (F. M. Vaz and R. J. A. Wandars, Biochem. J., 361, 417-429, 2002).

L-ascorbic acid may be a principle co-factor in the metabolism of L-carnitine. It has been postulated and demonstrated that an experimental vitamin C deficiency resulted in increased urinary excretion of L-carnitine. This increased excretion of L-carnitine may be due to either decreased absorption from dietary sources, or increased excretion from the kidney. Several methods have been described to measure the concentration of L-carnitine biosynthesis metabolites in biological fluids and tissues.
The kidney plays a major role in L-carnitine biosynthesis, excretion and acylation. Unlike in the rat, human kidney contains the enzymes needed to form L-carnitine from N-6 trimethyl-L-lysine (K, Doqi, National Kidney Foundation. Am. J. Kidney Dis., 35, 6 Suppl 2 S1-140, 2000). This L-carnitine precursor, TML, is found to be increased in plasma of patients with chronic renal failure. Free L-carnitine formed in the kidney as well as L-carnitine reabsorbed from the glomerular filtrate may be acylated in the proximal tubule. Isolated rat cortical tubule suspensions contain total L-carnitine concentrations of 2.85 micromols/g protein. During incubation over 60 min, the acylcarnitine/carnitine ratio decreased, indicating deacylation of acylcarnitine in proximal tubules. Exogenous L-carnitine was acylated at a rate of 35 micromols/h/g protein. Besides pyruvate and acetate, ketone bodies stimulated the acylation rate several fold, indicating that these substrates are a major source of acetyl-CoA for the acylation reaction. This may explain the higher acetylcarnitine/L-carnitine ratio found in urine under ketotic conditions.
However, later data shows that the brain participates in active synthesis of L-carnitine from TML takes place (F. M. Vaz and R. J. A. Wandars, Biochem. J., 361, 417-429, 2002). The concentration of butyrobetaine in plasma and tissues was determined by isolating butyrobetaine via HPLC or ion-exchange chromatography, and using BBD to convert it into L-carnitine. In humans, the level of butyrobetaine in urine is low (about 0.3 micromole/mmole L-creatinine) compared with the concentrations in plasma (4.8 mmole & 1.8 mmole).
The concentration of L-carnitine in plasma from both humans and rats is age and sex dependent. In humans, the plasma L-carnitine concentration increases during first year of life (from about 0.15 to about 0.40 mmole) and remains the same for both sexes until puberty. From puberty to adulthood, plasma L-carnitine concentrations in males increases and stabilizes at a level that is significantly higher than those in females (50 micromole compared to 40 micromole).
Obviously, carnitine is available from exogenous sources (meat, milk). However, work has been done to see if exogenous carnitine would ameliorate the symptoms of Juvenile Neuronal Ceroid Lipofuscinosis, not LINCL. This research in dogs showed that it made the dogs more functional and they lived 10% longer than untreated dogs, but the dogs still died very young compared to unaffected dogs and brain glucose hypometabolism and cerebral atrophy were not reduced (Siakotis, Katz et al., European Journal Pediatric Neurology 5, (Suppl. A): 151-156, 2001). It is an exciting prospect to see that TML may indeed be that therapeutic agent to cause positive brain metabolism based upon the results we have seen.
This invention provides a method of exogenic supplementation with TML to affect L-carnitine biosynthesis, thereby influencing ATP levels.